用碳-铂复型与免疫电镜技术对人脑胶质瘤细胞系BT_(325)整装细胞骨架的研究

STUDY OF WHOLE-MOUNT CYTOSKELETON OF HUMAN GLIOBLASTOMA BT_(325) CELLS BY CARBONPLATINUM COATING AND IMMUNOELECTRON MICROSCOPY

人脑胶质瘤细胞系BT_(325)细胞用含Triton X-100的缓冲液处理后,用针对波形纤维蛋白(vimentin)的单克隆抗体,与胶质纤维酸性蛋白(GFAP)抗血清进行间接免疫荧光染色。在荧光显微镜下观察后,将细胞用酒精逐级脱水,然后进行临界点干燥。将干燥后的细胞进行碳-铂喷镀,所得到的碳-铂复型用透射电镜观察。用本法可清晰地观察到完整的细胞骨架,以及微管、微丝与中间丝的形态与分布。在经vimentin单克隆抗体免疫染色的细胞内,中间丝直径较粗;在用GFAP抗血清染色后,只有部分细胞内中间丝直径较粗。如将Triten处理的细胞先用甲醛固定后,再进行免疫染色,中间丝直径增加较少;但如果先进行免疫染色后再用甲醛固定,中间丝直径显著增加。这与免疫荧光观察结果相一致,说明甲醛固定对中间丝分子上的抗原位点起破坏或遮蔽作用。

Cells of BT_(325), a human glioblastoma cell line, were treated with 0.5% Triton X-100 and immunostained with a monoclonal antibody to vimentin and antiserum to glial fibrillary acidic protein (GFAP), and followed by fluorescence labeled secondary antibody. After observation with fluorescence microscope the cells were dehydrated in increasing grades of ethanol and then critical point-dried. The carbon-platinum coating replicas of cytoskeleton were observed by transmission electron microscopy. The morphology of whole cytoskeleton as well as the arrangement of microtubules, microfilaments and intermediate filaments were clearly observed by this technique. It was observed that the diameter of intermediate filaments highly increased in the cells stained with the monoclonal antibody to vimentin. In the samples stained with anti-GFAP intermediate filaments were decorated in some cells but undecorated in other cells. The results indicates that all BT_(325) cells express vimentin, but only a fraction of the cells express GFAP. It was also observed that if the cells were fixed with formaldehyde before immunostaing with the monoclonal antibody to vimentin, the diameter of the intermediate filaments only slightly increased. However, the diameter of the intermediate filaments highly increaed in the cells stained firstly with the monoclonal antibody to vimentin and followed by formaldehyde fixation. This result is in accord with that of our previous immunofluorescence study. It indicates that formaldehyde fixation may 'mask' certain epitopes on vimentin molecules.